Myristoylated Naked2 escorts transforming growth factor α to the basolateral plasma membrane of polarized epithelial cells

Abstract

The epidermal growth factor receptor ligands transforming growth factor ␣ (TGF␣) and amphiregulin are delivered to the basolateral surface of polarized epithelial cells where they are cleaved by TACE͞ ADAM17. Basolateral sorting information resides in their cytoplasmic tail domains, but tail-interacting proteins required for basolateral trafficking have not been identified. Naked (NKD)1 and NKD2 are mammalian homologs of Drosophila Naked Cuticle, which negatively regulates canonical Wnt signaling by binding Dishevelled. We present evidence that NKD2, but not NKD1, binds to basolateral sorting motifs in the cytoplasmic tail of TGF␣. Processing and cell-surface delivery of TGF␣ are accelerated in NKD2-overexpressing Madin-Darby canine kidney cells. NKD2 is myristoylated on glycine, the second residue. On expression of myristoylation-defective (G2A) NKD2, neither NKD2 nor TGF␣ appears at the basolateral plasma membrane of polarized Madin-Darby canine kidney cells; however, membrane staining for TGF␣ is restored on silencing expression of this mutant NKD2. Amphiregulin does not interact with NKD2 and retains its basolateral localization in G2A-NKD2-expressing cells, as do Na ؉ , K ؉ ATPase ␣1 and E-cadherin. These data identify an unexpected function for NKD2, i.e., myristoylation-dependent escort of TGF␣ to the basolateral plasma membrane of polarized epithelial cells.

Naked2 Acts as a Cargo Recognition and Targeting Protein to Ensure Proper Delivery and Fusion of TGF-α–containing Exocytic Vesicles at the Lower Lateral Membrane of Polarized MDCK Cells

“…We recently reported that Naked2 binds to these domains but not to TGF␣'s PDZ recognition domain (Li et al, 2004 There are four regions conserved between Naked1 and Naked2 ( Figure 1A). The most conserved region among Naked family members is Naked homologous region 1 (NHR1), residues 107-175 in human Naked2, which contains an EF-hand motif.…”

“…We previously observed that myristoylation-deficient G2A Naked2-associated vesicles accumulate asymmetrically at the basolateral corner of polarized MDCK cells (Li et al, 2004). To observe the site of docking and fusion of wild-type Naked2-associated vesicles in polarized MDCK cells, we used live cell microscopy.…”

“…We recently reported that Naked2 binds to these domains but not to TGF␣'s PDZ recognition domain (Li et al, 2004). Naked1 and 2 are the two mammalian orthologues of Naked Cuticle, an inducible negative regulator of canonical Wnt signaling as determined by genetic analysis in Drosophila (Rousset et al, 2001;Yan et al, This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E07-02-0172) on June 6, 2007.…”

“…Glycine, the second residue, undergoes myristoylation. Naked homologue region 1 (NHR1) consists of residues 107-175; NHR2 includes residues 236-265; TGF␣ tail-binding domain (TTB) was mapped to residues 300-385 (Li et al, 2004), and H16 represents the last 22 residues that contain 16 histidines. (B) Live cell microscopy of a typical homotypic vesicle-vesicle fusion event.…”

“…In myristoylation-deficient G2A Naked2-expressing Madin-Darby canine kidney (MDCK) cells, Naked2 vesicles accumulate at a basolateral corner of the cell, and TGF␣ is unable to reach the plasma membrane. These effects appear to be specific for TGF␣ because amphiregulin, another basolaterally targeted EGFR ligand, is found at the cell surface of both wild-type and myristoylation-deficient G2A Naked2-expressing MDCK cells (Li et al, 2004). The present studies were designed to determine how Naked2 delivers these TGF␣-containing vesicles to their proper destination.…”